Fig 8.

FOSL1 regulates the integrin αv and β3 promoters indirectly through SP1. (A) Chromatin immunoprecipitation of FOSL1 on αv and β3 and NOTCH4 promoters. FOSL1 silencing (shFOSL1) significantly reduced the signal on all three promoters. Primer positions for the amplification of αv and β3 promoters are reported in Fig. 7A. Amplification of the NOTCH4 promoter region, which is bound by both FOSL1 (directly through an AP-1 binding site) and SP1, was used as a control. (B) Chromatin immunoprecipitation of SP1 on αv and β3 and NOTCH4 promoters. Cell treatment with mithramycin A for 2 h strongly inhibited SP1 binding to all three promoters. (C) Chromatin immunoprecipitation of FOSL1 on αv and β3 and NOTCH4 promoters in cells untreated (Vehicle) or treated with mithramycin A for 2 h. Mithramycin A inhibited the binding of FOSL1 on the on αv and β3 promoters but not the NOTCH4 promoter where FOSL1 binds directly to an AP-1 site. (D) Nuclear extracts obtained from HUVEC were either immunostained (Input) or subjected to immunoprecipitation (IP) with anti-FOSL1 or anti-SP1 antibodies as indicated. (E) Chromatin immunoprecipitation of JunD on αv and β3 promoters. FOSL1 silencing significantly reduced the JunD association on both promoters. (F and G) Chromatin immunoprecipitation analysis of histone modifications on αv and β3 promoters in control cells and in FOSL1-silenced cells. FOSL1 silencing induces a significant increase of H4ac at K16. Mean values from three independent experiments are shown with standard deviations.